1.Skeleton Binding Protein 1 of Plasmodium berghei Influences Deformability and Cytoskeletal Ultrastructure of Infected Erythrocyte
Xin-Yue GUO ; Huan-Qi ZHAO ; Yan-Xuan ZHONG ; Ru-Meng JIANG ; Yao-Xian LI ; Lei-Ting PAN ; Qian WANG ; Xiao-Yu SHI
Progress in Biochemistry and Biophysics 2026;53(4):1015-1027
ObjectiveThe malaria parasites remodel the host erythrocyte structure by exporting parasite proteins that interact with the membrane skeleton proteins of red blood cells (RBCs), facilitating their intracellular survival and pathogenicity. Skeleton-binding protein 1 (SBP1) is a conserved exported protein across Plasmodium species. In Plasmodium falciparum, SBP1 has been reported to interact with erythrocyte membrane skeleton proteins 4.1R and spectrin, while its contribution to erythrocyte remodeling and parasite virulence in Plasmodium berghei (Pb) remains unclear. This study aims to determine whether PbSBP1 associates with the host cytoskeletal protein 4.1R and to investigate its role in the remodeling of host RBCs and the pathogenicity of Plasmodium berghei. MethodsIn Plasmodium berghei, the relationship between PbSBP1 and the erythrocyte cytoskeletal protein 4.1R was examined using co-immunoprecipitation. A Pbsbp1 gene knockout mutant of Plasmodium berghei (Pbsbp1∆) was generated based on the principle of double crossover homologous recombination. The deformability of erythrocytes infected with Pbsbp1∆ parasites was assessed using microfluidic methods. Microchannels with an array of cylindrical pillars were used to detect modifications in infected RBC deformability. The infected RBCs were squashed between the rows and recovered between the columns and the transit velocity (μm/s) of infected RBCs travelling through the microchannel was recorded. The component of the erythrocyte membrane skeleton junctional complex, tropomodulin (TMOD), was fluorescently labeled, and the cytoskeletal network of infected erythrocytes was imaged using super-resolution stochastic optical reconstruction microscopy (STORM) to analyze ultrastructural changes in the cytoskeleton of wild-type (WT) and Pbsbp1∆-infected erythrocytes. Actin-based junctional complexes were displayed as individual clusters by the labeled TMOD in the STORM images, and the cluster densities and distances between adjacent clusters of infected RBCs were calculated. Additionally, rodent malaria models (BALB/c mice) and experimental cerebral malaria models (C57BL/6 mice) were employed to monitor the growth of Pbsbp1∆ and WT parasites during the intraerythrocytic stage and their capacity to induce cerebral malaria in mice. ResultsPbSBP1 may participate in the remodeling of infected erythrocytes through direct or indirect interaction with the erythrocyte cytoskeletal protein 4.1R. Microfluidic assays revealed that the deformability of erythrocytes infected with Pbsbp1∆ parasites was significantly enhanced compared to those infected with WT parasites. STORM imaging further demonstrated that the ultrastructure of the erythrocyte cytoskeleton in Pbsbp1∆-infected cells was altered relative to that in WT-infected erythrocytes. The distances between nearest neighbors of clusters had a tendency to increase while the cluster densities were decreased in Pbsbp1∆-infected RBCs compared to WT-infected RBCs. Subsequent phenotypic analysis indicated that the growth rate of Pbsbp1∆ parasites during the intraerythrocytic stage was significantly slower than that of WT parasites, and their ability to induce cerebral malaria in mice was also attenuated. These findings suggest that PbSBP1 is involved in the remodeling of the erythrocyte membrane skeleton, likely through its direct or indirect interaction with protein 4.1R, thereby regulating the deformability of infected erythrocytes and influencing the pathogenicity of the blood-stage parasites. ConclusionThis study establishes a role for PbSBP1 in host erythrocyte remodeling and parasite virulence, providing new research strategies for the prevention and treatment of malaria.
2.Effects of subanesthetic dose of esketamine on postoperative anxiety and recovery in patients undergoing laparo-scopic cholecystectomy
Zhangzhen ZHONG ; Xian ZHENG ; Ting XU ; Jie WANG ; Hui CAO ; Xinggen ZHOU ; Hui LI ; Jiacheng ZHAO ; Hui LIU ; Chao ZHANG
China Pharmacy 2026;37(2):204-209
OBJECTIVE To investigate the effects of subanesthetic dose of esketamine on postoperative anxiety and recovery in patients undergoing laparoscopic cholecystectomy. METHODS A total of 200 patients scheduled for laparoscopic cholecystectomy at Suzhou Ninth Hospital Affiliated to Soochow University from January 2023 to December 2024 were randomly assigned to control group (n=100) and observation group (n=100). One minute before the initiation of anesthesia, patients in the control group received intravenous injections of Propofol emulsion injection, Sufentanil citrate injection, and Succinylcholine chloride injection. On this basis, patients in the observation group received an intravenous injection of Esketamine hydrochloride injection. The anxiety status of patients in both groups was compared, along with their general intraoperative conditions (including sufentanil dosage, duration of pneumoperitoneum, operative time, anesthesia time, and extubation time), postoperative recovery, incidence of adverse reactions, and the need for dezocine rescue analgesia. Heart rate and mean arterial pressure, entropy index (state entropy and response entropy), inflammatory marker levels [interleukin-6 (IL-6) and C-reactive protein (CRP)], numerical rating scale (NRS) for pain intensity were compared between the two groups at different time points. RESULTS No significant differences were found between the two groups in pneumoperitoneum duration, operative time, anesthesia time,extubation time, incidence of postoperative dry mouth, entropy index or length of stay in the post-anesthesia care unit (P>0.05). Compared with the control group, the observation group showed significantly lower postoperative STAI-S scores, reduced intraoperative sufentanil consumption, decreased incidence of postoperative nausea, vomiting, and shivering, the need for dezocine rescue analgesia, as well as lower plasma IL-6 and CRP levels at 24 h after surgery, and NRS (P<0.05). The heart rate and mean arterial pressure of patients in the observation group at the start of surgery, end of surgery, and during extubation were all significantly higher than those in the control group (P<0.05). CONCLUSIONS Subanesthetic dose of esketamine can effectively alleviate postoperative anxiety, reduce intraoperative opioid consumption, suppress postoperative inflammatory response, relieve postoperative pain, and promote recovery in patients undergoing laparoscopic cholecystectomy.
3.Ameliorating Effect of Yifei Tongluo Prescription on Bleomycin-induced Pulmonary Fibrosis in Rats via Regulating NLRP3/Caspase-1/GSDMD Signaling Pathway and Epithelial-mesenchymal Transition
Bowen ZHOU ; Zefeng LI ; Xian MA ; Xuannian LI ; Jingwen WANG ; Fei XU ; Huaman LIU ; Xinhua JIA
Chinese Journal of Experimental Traditional Medical Formulae 2026;32(1):150-159
ObjectiveTo observe the effects of Yifei Tongluo prescription on the NOD-like receptor protein 3 (NLRP3)/Caspase-1/gasdermin D (GSDMD) pathway and epithelial-mesenchymal transition (EMT) in rats with pulmonary fibrosis. MethodsTracheal instillation of bleomycin was conducted to establish a rat model of pulmonary fibrosis. Thirty Sprague-Dawley (SD) rats were randomly divided into a blank group, a model group, a prednisone acetate group (1.17 mg·kg-1), and low- and high-dose Yifei Tongluo prescription groups (10.62 and 21.24 g·kg-1, respectively). Administration started on the 7th day after modeling, once a day for 28 consecutive days. The lung coefficient of each group was calculated. The pathological changes of lung tissues in each group were observed by hematoxylin-eosin (HE) staining and Masson staining. The expression of α-smooth muscle actin (α-SMA) and vimentin in rat lung tissues was detected by immunohistochemistry. The expression of NLRP3 inflammasome, E-cadherin (E-cad), and typeⅠ collagen (ColⅠ) in lung tissues was detected by immunofluorescence. The content of hydroxyproline (HYP), tumor necrosis factor (TNF)-α, interleukin (IL)-18, and IL-1β in rat serum was detected by enzyme-linked immunosorbent assay (ELISA). The mRNA expression levels of NLRP3, apoptosis-associated speck-like protein containing a CARD (ASC), IL-1β, and transforming growth factor (TGF)-β1 in rat lung tissues were determined by real-time quantitative polymerase chain reaction (Real-time PCR). The protein expression levels of NLRP3, GSDMD, ASC, and Caspase-1 in rat lung tissues were determined by Western blot. ResultsCompared with the blank group, the model group exhibited a significantly increased lung coefficient (P<0.01) and significantly increased range of pulmonary interstitial inflammation and collagen deposition. In addition, the levels of α-SMA, Vimentin, E-cad, and ColⅠ in lung tissues were significantly increased (P<0.01). The levels of fibrosis- and inflammation-related factors HYP, TNF-α, IL-18, and IL-1β in serum were significantly upregulated (P<0.01). The levels of factors related to the activation of NLRP3 inflammasome in lung tissues, including NLRP3, GSDMD, ASC, Caspase-1, IL-1β, and TGF-β1, were significantly upregulated (P<0.01). Compared with the model group, the Yifei Tongluo prescription groups showed improved lung coefficients. Additionally, the extent of lung inflammation and collagen deposition was significantly reduced. The expression of α-SMA, Vimentin, E-cad, and ColⅠ in lung tissue was significantly decreased (P<0.01). The levels of HYP, TNF-α, IL-18, and IL-1β in serum were significantly reduced (P<0.01). The expression levels of NLRP3, GSDMD, ASC, Caspase-1, IL-1β, and TGF-β1 in lung tissue were also significantly decreased (P<0.01). ConclusionYifei Tongluo prescription can regulate the NLRP3/Caspase-1/GSDMD pathway, down-regulate release of pro-inflammatory and pro-fibrotic cytokines, alleviate NLRP3 inflammasome-mediated pyroptosis and EMT, and thereby improve pulmonary fibrosis in rats.
4.Bibliometric and visual analysis of the research status and trends of senescence in osteoporosis
Haiwen ZHANG ; Xian ZHANG ; Taichuan XU ; Chao LI
Chinese Journal of Tissue Engineering Research 2026;30(6):1580-1591
BACKGROUND:Cellular senescence,which triggers the aging process of physiological decline in the body,is closely related to osteoporosis.With the development of aging research,significant progress has been made in the study of targeted clearance of senescent cells.Accordingly,senolytic cell therapy for osteoporosis based on this has attracted increasing attention.Current research on the association between aging and osteoporosis shows the characteristic of multidisciplinary intersection.However,existing reviews are mostly based on a single database(such as PubMed)and lack a systematic comparative analysis of Chinese and English literature.Therefore,it is of great academic value to integrate resources from multiple databases and systematically reveal the research status and hot trends of aging in the field of osteoporosis research.OBJECTIVE:To summarize the development,current status,hotspots,and future trends of research on aging in the field of osteoporosis over the past 20 years,so as to provide references for future related research.METHODS:We searched for research literature on the correlation between aging and osteoporosis in CNKI,WanFang,VIP and Web of Science Core Database.The search time span was from August 1,2004 to September 24,2024.Then,we used NoteExpress 4.0 for data cleaning and CiteSpace 6.3R1(Advanced)and Excel(2024)for literature analysis.RESULTS AND CONCLUSION:Since 2004,research on the correlation between aging and osteoporosis has shown a significant growth trend.Bibliometric analysis shows that(as of September 2024),and 1 275 English documents and 151 Chinese documents have been published,with the highest number of publications in China and the United States.In terms of publishing institutions,the Mayo Clinic ranked first,followed by Shanghai Jiao Tong University and the University of California system.As for core authors,Sundeep Khosla and Joshua Nicholas Farr were the authors with the most publications and citations,while Pei Lingpeng and Hui Bodi were the most worthy of attention in Chinese literature.After the keywords"aging"and"osteoporosis"and their synonyms were excluded,it was found that cellular senescence,senescence-associated secretory phenotype,signaling pathways,and targeted senolytic cell clearance therapy haven been the current research hotspots and theoretical frontiers.
5.Bibliometric and visual analysis of the research status and trends of senescence in osteoporosis
Haiwen ZHANG ; Xian ZHANG ; Taichuan XU ; Chao LI
Chinese Journal of Tissue Engineering Research 2026;30(6):1580-1591
BACKGROUND:Cellular senescence,which triggers the aging process of physiological decline in the body,is closely related to osteoporosis.With the development of aging research,significant progress has been made in the study of targeted clearance of senescent cells.Accordingly,senolytic cell therapy for osteoporosis based on this has attracted increasing attention.Current research on the association between aging and osteoporosis shows the characteristic of multidisciplinary intersection.However,existing reviews are mostly based on a single database(such as PubMed)and lack a systematic comparative analysis of Chinese and English literature.Therefore,it is of great academic value to integrate resources from multiple databases and systematically reveal the research status and hot trends of aging in the field of osteoporosis research.OBJECTIVE:To summarize the development,current status,hotspots,and future trends of research on aging in the field of osteoporosis over the past 20 years,so as to provide references for future related research.METHODS:We searched for research literature on the correlation between aging and osteoporosis in CNKI,WanFang,VIP and Web of Science Core Database.The search time span was from August 1,2004 to September 24,2024.Then,we used NoteExpress 4.0 for data cleaning and CiteSpace 6.3R1(Advanced)and Excel(2024)for literature analysis.RESULTS AND CONCLUSION:Since 2004,research on the correlation between aging and osteoporosis has shown a significant growth trend.Bibliometric analysis shows that(as of September 2024),and 1 275 English documents and 151 Chinese documents have been published,with the highest number of publications in China and the United States.In terms of publishing institutions,the Mayo Clinic ranked first,followed by Shanghai Jiao Tong University and the University of California system.As for core authors,Sundeep Khosla and Joshua Nicholas Farr were the authors with the most publications and citations,while Pei Lingpeng and Hui Bodi were the most worthy of attention in Chinese literature.After the keywords"aging"and"osteoporosis"and their synonyms were excluded,it was found that cellular senescence,senescence-associated secretory phenotype,signaling pathways,and targeted senolytic cell clearance therapy haven been the current research hotspots and theoretical frontiers.
6.Analysis of Quality Changes of Small Packaged Alismatis Rhizoma Decoction Pieces Under Different Packaging and Storage Conditions
Gaoting YANG ; Rui XIAN ; Zimin WANG ; Zongyi ZHAO ; Zhiqiong LAN ; Xiaoli PAN ; Min LI
Chinese Journal of Experimental Traditional Medical Formulae 2026;32(2):179-188
ObjectiveTo screen suitable packaging and storage conditions for small packaged Alismatis Rhizoma decoction pieces, laying the foundation for developing standardized storage, maintenance techniques and determining shelf life. MethodsUsing the accelerated stability test method, the small packaged decoction pieces of Alismatis Rhizoma were placed in polyethylene plastic bags, aluminum foil polyethylene composite bags, and cowhide coated paper bags under temperature of (40±2) ℃ and relative humidity of (75±5)% conditions, the quality testing was conducted at the end of the 0th, 1st, 2nd, 3rd, and 6th month, respectively. Using long-term stability test method, an orthogonal experiment was designed to investigate storage conditions, packaging materials, and packaging methods. At the end of the 0th, 1st, 3rd, 6th, 9th, 12th, 18th, and 24th month, the quality of small packaged Alismatis Rhizoma decoction pieces was tested under different packaging and storage conditions(including 2 packaging methods:vacuum packaging and sealed packaging, 3 storage conditions:room temperature, cool, and modified atmosphere, 3 packaging materials:cowhide coated paper bag, aluminum foil polyethylene composite bag, and polyethylene plastic bag). Then, the G1-entropy weight method combined with orthogonal experiment was used to analyze the quality changes of the decoction pieces under different packaging and storage conditions to identify optimal packaging and storage conditions. The quality testing indicators for Alismatis Rhizoma decoction pieces were expanded beyond those specified in the 2020 edition of the Pharmacopoeia of the People's Republic of China. In addition to the existing indicators(characteristics, moisture content, extractives, and the total content of 23-acetyl alisol B and 23-acetyl alisol C), new indicators including color value, water activity, total triterpenoid content, and alisol B content have been added. ResultsThe accelerated stability test results indicated that the quality of small packaged Alismatis Rhizoma decoction pieces was more stable when packaged in aluminum foil-polyethylene composite materials compared to cowhide-coated paper bags and polyethylene plastic bags. Analysis of the long-term stability test results using the G1-entropy weight method combined with orthogonal experiment revealed that storage conditions had the greatest impact on both raw and salt-processed products, followed by packaging materials, while the packaging method had the least influence. For both types of small packaged Alismatis Rhizoma decoction pieces, modified atmosphere storage demonstrated superior efficacy compared to cool storage or room temperature storage. Storage in aluminum foil-polyethylene composite bags was superior to polyethylene plastic bags or cowhide-coated paper bags. However, the stability of sealed raw products was better than vacuum-packed ones, whereas vacuum-packed salt-processed products exhibited greater stability than their sealed counterparts. ConclusionBased on the results of the quality changes of small packaged Alismatis Rhizoma decoction pieces under different storage conditions, it is recommended that the suitable storage packaging conditions for small packaged raw products are sealed packaging with aluminum foil polyethylene composite bags and controlled atmosphere storage, and the suitable storage and packaging conditions for small packaged salt-processed products are vacuum packaging with aluminum foil polyethylene composite bags and controlled atmosphere storage.
7.Factors influencing daily blood donation attendance and their spatial heterogeneity at fixed donation sites in Nanjing
Zhanghui LI ; Wenbiao LIANG ; Wenjia HU ; Xian CHEN ; Wenqing YUAN ; Nizhen JIANG
Chinese Journal of Blood Transfusion 2026;39(7):908-916
Objective: To analyze the time-related factors influencing daily blood donation attendance at fixed donation sites in Nanjing and the heterogeneity among sites, and to provide evidence for optimizing site layout and operational management. Methods: Based on daily attendance data from 17 fixed donation sites in Nanjing for the full year of 2025, matched with contemporaneous meteorological and air quality data, a two-stage negative binomial generalized linear mixed model (NB-GLMM) was constructed. In the second stage, site-level static characteristics (number of scenic spots, daily operating hours, number of shopping malls, public transport stops, hospitals, universities, and residential buildings) were additionally incorporated. Model comparison was performed using Akaike information criterion (AIC). Significance of random effects was tested by likelihood ratio test, and site-specific random effects were extracted using empirical Bayes estimation. Robustness of the results was verified through residual diagnostics and multiple sensitivity analyses. Results: The first-stage model showed that rainfall was associated with lower attendance (IRR=0.943, P<0.001); holidays were associated with higher attendance (IRR=1.162, P<0.001); Saturdays and Sundays showed higher attendance than Mondays, with the strongest association on Sundays (IRR=1.195, P<0.001). Temperature exhibited a non-linear relationship (linear term P=0.051, quadratic term P=0.036). The joint results of the linear and quadratic terms for temperature suggested a nonlinear relationship between attendance and temperature. After introducing static characteristics in the second stage, the number of scenic spots (IRR=1.372, P=0.008) and daily operating hours (IRR=1.823, P<0.001) were found to be positively associated with attendance, while the numbers of shopping malls, public transport stops, hospitals, universities, and residential buildings showed no significant effects. Conclusion: Daily blood donation attendance at fixed sites in Nanjing is influenced by multiple factors including weather, day of the week, holidays, and site surroundings. Substantial heterogeneity exists among different sites; extended operating hours and more nearby scenic spots are associated with increased attendance.
8.Impact of wearable devices in assisting disease monitoring on heart-focused anxiety in patients with tachyarrhythmias
Yuying LI ; Xinyue DONG ; Zhiyun SHEN ; Xian ZHANG ; Caiying YANG ; Yuxuan HUANG ; Meiqiong YAN
Chinese Journal of Clinical Medicine 2026;33(3):445-451
Objective To explore the impact of wearable devices in assisting disease monitoring on heart-focused anxiety in patients with tachyarrhythmias. Methods A cross-sectional study design was conducted. 305 patients with tachyarrhythmias who attended the department of cardiology of Zhongshan Hospital, Fudan University in Shanghai from July 2025 to December 2025 were enrolled as the research subjects, and clinical data of patients were collected. Univariate analysis and multiple stepwise regression analysis were used to analyze the impact of wearable devices in assisting disease monitoring on patients’ heart-focused anxiety. Results The mean score of heart-focused anxiety in the 305 patients with tachyarrhythmias was 33.16±9.70. Among them, 104(34.10%) patients used wearable devices in assisting disease monitoring. The results of univariate analysis and multiple stepwise regression showed that the use of wearable devices in assisting disease monitoring was an influencing factor for heart-focused anxiety (P<0.05), and the patients who used wearable devices in assisting disease monitoring had a significantly lower level of heart-focused anxiety (P<0.05). Conclusions The use of wearable devices in assisting disease monitoring is associated with a lower level of heart-focused anxiety in patients with tachyarrhythmias. Medical staff can guide patients to rationally use wearable devices in assisting disease monitoring on the basis of evaluating their needs and device operation capabilities.
9.Chinese expert consensus on salvage esophagectomy for esophageal cancer after definitive chemoradiotherapy
Zhaoxian LIN ; Yang HU ; Lei XIAN ; Yun LI ; Jinbo ZHAO ; Xiaobin HOU ; Shuangping ZHANG ; Sunkui KE ; Changying GUO ; Songping XIE ; Haitao WEI ; Yong LI
Chinese Journal of Clinical Thoracic and Cardiovascular Surgery 2026;33(07):977-987
Definitive chemoradiotherapy (dCRT) has become a cornerstone in the treatment of locally advanced esophageal cancer; however, local control remains suboptimal, and persistent lesions or locoregional recurrences after treatment are not uncommon. For patients without distant metastases but with local failure, whether surgical intervention can still offer curative potential remains a major clinical dilemma. Salvage esophagectomy (SE) offers potential long-term survival for selected patients, but this procedure is performed in the context of severe fibrosis, impaired local blood supply, and obscured anatomical planes following chemoradiotherapy, resulting in significantly higher perioperative risk compared to primary esophagectomy. Consequently, controversies exist regarding patient selection, preoperative restaging, choice of surgical approach, extent of lymphadenectomy, gastrointestinal reconstruction, and perioperative management. In recent years, with the refinement of restaging modalities such as PET/CT, the accumulation of experience in high-volume centers, and emerging evidence from clinical studies, the clinical role of SE has gradually shifted from a "high-risk salvage measure" to a "selective curative strategy aimed at achieving long-term survival in carefully selected patients". Nevertheless, standardized guidelines for patient selection, technical approaches, and perioperative management are still lacking. Based on current evidence and clinical experience, experts organized by the Integrated Esophageal Cancer Committee of Chinese Anti-Cancer Association systematically reviewed key issues regarding SE, including its definition, indications, preoperative evaluation, choice of surgical approach, lymphadenectomy, gastrointestinal reconstruction, and perioperative management, and formulated a Chinese expert consensus. This consensus aims to provide guidance for standardized assessment, appropriate referral, individualized surgical decision-making, and optimized perioperative management of patients with locoregional failure after dCRT. Ultimately, this will increase the likelihood of R0 resection, reduce the risk of severe complications, and promote the safer, more judicious, and standardized implementation of SE in high-risk scenarios.
10.The Neural Circuit Characteristics of Repetitive Transcranial Magnetic Stimulation Over The Dorsolateral Prefrontal Cortex for The Treatment of Migraine
Chen-Xia JIN ; Bo-Lin TAN ; Yang YE ; Ji-Qing HE ; Ling-Yan WANG ; Zhong-Ming GAO ; Yu-Jun WANG ; Hui-Li LIU ; Yong-Xing YAN ; Xian-Wei CHE
Progress in Biochemistry and Biophysics 2026;53(7):1953-1968
ObjectiveMigraine is a leading neurological disorder and the fourth most common cause of years lived with disability worldwide, affecting nearly 116 million individuals. Although pharmacological treatments are available, their efficacy is often limited by side effects and variable response rates. Repetitive transcranial magnetic stimulation (rTMS) over the dorsolateral prefrontal cortex (DLPFC) offers a safe, non-invasive alternative for migraine management. However, the neurophysiological mechanisms, particularly how rTMS modulates local cortical excitability and distributed pain-related circuits, remain poorly understood. Elucidating these mechanisms is essential for optimizing treatment protocols and improving clinical outcomes. MethodsThis study employed concurrent transcranial magnetic stimulation and electroencephalography (TMS-EEG) to investigate neuroplastic and neurocircuitry mechanisms of DLPFC-rTMS in migraine. Study 1 compared 30 migraineurs and 28 healthy controls to identify abnormalities in TMS-evoked potentials (TEPs) and significant current density (SCD) within sensory-discriminative regions including the primary somatosensory cortex (S1) and posterior insula (pINS), cognitive-affective regions including the anterior insula (aINS) and midcingulate cortex (MCC), and a descending modulatory region, the periaqueductal gray (PAG). Study 2 used a single-blind, crossover, sham-controlled design in 34 healthy participants. Each participant received both active (10 Hz, 80% RMT, 1 500 pulses) and sham DLPFC-rTMS in counterbalanced order. TMS-EEG and cold pain tolerance were assessed before and after each session. ResultsIn Study 1, migraineurs showed a significantly less negative N120 amplitude compared to healthy controls (P=0.027, Cohen’s d=0.60), indicating local intracortical disinhibition. No group differences were observed for N40, P60, or P180 components. At the source level, migraineurs exhibited significantly higher SCD in the S1, pINS, aINS, and MCC (allQ<0.05), but not in the ventroposterior thalamus (vpTHAL), mediodorsal thalamus (mdTHAL), or PAG. In Study 2, active rTMS significantly reduced SCD from pre- to post-stimulation in the S1, aINS, and MCC (all Q<0.05). Sham stimulation also reduced SCD in the S1 (Q<0.05) but not in the aINS or MCC. Although no significant group-level analgesic effect was observed between active and sham conditions (P=0.107), correlation analyses revealed that greater SCD reductions in the S1 and MCC were significantly associated with higher post-rTMS pain tolerance (R=-0.487 and -0.495, both Q<0.01) and larger improvements in pain tolerance(R=-0.487 and -0.451, both Q<0.05). No such correlations were found following sham stimulation, suggesting that the behavioural relevance of neural changes is specific to active rTMS. ConclusionThis study provides novel evidence that migraineurs exhibit both local neuroplastic abnormalities (reduced N120 amplitude) and hyperactivity in key pain-processing regions (S1, pINS, aINS, MCC). A single session of DLPFC-rTMS reduced hyperactivity in the aINS, MCC, and S1. Notably, greater reductions in the S1 and MCC were associated with improved pain tolerance. These findings identify distinct cortical circuitries, particularly within the cognitive-affective pain network, that may serve as potential biomarkers for optimizing rTMS treatment in migraine and other chronic pain conditions. Future studies should validate these results in patient populations experiencing spontaneous migraine attacks and explore multi-session or accelerated rTMS protocols.

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